As double stranded DNA (dsDNA) is heated it dissociates or melts, that is the two strands separate. The melting temperature may, for example, be in the range 60-90° C. The melting temperature (Tm) depends upon a range of factors including, by way of example, GC-content (guanine-cytosine content), the length of the DNA strand (or amplicon length), the degree of methylation, and secondary and tertiary structure. Further, where there is a mismatch between a probe and a target sequence, for example due to the presence of a SNP (single nucleotide polymorphism) then the melting temperature will differ compared with when there is no mismatch. Thus, for example, it can be useful to detect changes in melting temperature of DNA-hybridisation between a specific oligonucleotide probe and a gene target when a SNP is present. Melting point analysis is often used in conjunction with PCR (polymerase chain reaction) amplification, for example to amplify somatic cell mutations which may only be present in a very few copies in a sample. More particularly in real time quantitative PCR (qPCR) melt/dissociation curve analysis can be used to determine the number and/or approximate size of PCR products. Thus a melt curve may have one or more peaks depending upon the product or products present (the identities of products may be determined by fractionating the product(s) on a gel). According to one definition the melting temperature, Tm, may be defined as the temperature at which 50% of the DNA sample is double stranded and 50% of the sample is single stranded.
Typically a melt curve is determined by measuring the change in fluorescence of a double stranded DNA sample with temperature, more particularly by observing the reduction in fluorescence due to melting of the dsDNA. In one approach a DNA-binding dye is employed. Typically such a dye is non-specific and binds to dsDNA, for example generated during PCR amplification; the dye is highly fluorescent when bound to dsDNA but exhibits only a low fluorescence when not bound. Thus there is a reduction in fluorescence when the dsDNA melts. Such dies typically operate by intercalation and/or minor groove binding; examples are SYBR™ Green and quencher-labelled primers. Alternatively sequence specific fluorescent DNA-based probes may be employed, typically containing a fluorescent element and a quenching element such that when the probe hybridises to the complementary target these elements are separated so that a fluorescent signal is generated.
FIG. 1a shows, schematically, a typical fluorescence melt curve with fluorescence (in arbitrary units) on the Y axis and temperature on the X axis. Initially the fluorescence exhibits a linear decrease with increasing temperature until a point A is reached at which the fluorescence drops below the level predicted by this linear decrease as the dsDNA begins to melt. Eventually, at point B, the melting is complete and the fluorescence level drops towards zero. A melt curve is often plotted as the negative first derivative of the fluorescence signal with respect to temperature (−dF/dT) as shown schematically in FIG. 1b. This facilitates determine the melting temperature, which correspondents to the peak of the derivative curve (the maximum slope of the fluorescence curve).
Although melt/dissociation curve analysis is typically performed by determining the derivative of the fluorescence signal with temperature this is not essential. For example in some other approaches melt curve analysis is performed directly on the fluorescence signal, which is typically then normalised amongst samples for comparison.
As shown in FIG. 2, in quantitative PCR there is phase during which the amount of target grows exponentially prior to some limiting point (linear on a semi-log plot). The amount of DNA initially present in a sample can be determined from the point at which fluorescence is first detected above the background level—this is referred to as the cycle number threshold (Ct) or crossing point. This is inversely correlated to the logarithm of the initial copy number. Initially the level of fluorescence from the amplified PCR product is too low to be detected but after a number of PCR cycles, depending upon the initial amount of sample DNA, the product is detectable. The sooner the accumulated product is detected, the lower the cycle number threshold, and the higher the initial amount of sample DNA. For accuracy, and to detect lower levels of sample DNA, it is desirable for the instrument to detect low levels of fluorescence signal (although the background level can drift).
Particularly in an instrument which is able to perform melt curve analysis, the desire for high sensitivity conflicts with the need to obtain accurate melt temperatures, which can be affected by saturation/limiting of the fluorescence detection at high signal levels.
We will describe techniques which address these and other problems associated with PCR, in particular qPCR, and melt/dissociation curve analysis.